Biocatalysts such as enzymes and microbial cells are useful for the production of fine chemicals, general-purpose chemicals, medical intermediates, and biofuels. Biocatalysts catalyze efficient and highly selective reaction under mild conditions such as normal temperature, normal pressure, and neutral conditions. However, bioprocess using biocatalysts requires high production costs, and this problem hinders its commercialization.
Immobilization of biocatalysts has been considered as an important strategy for the cost reduction of bioprocess, because it allows use of the catalyst in repeated and/or continuous reactions, simplifies recovery and isolation of the catalyst and product from the reactor, facilitates the regeneration of the catalyst, and allows the increase in the catalyst concentration per volume. In addition, the use of the whole cell catalyst using the whole of microbial cells markedly contributes to the cost reduction of bioprocess, because, for example, it dispenses with the isolation and purification of the enzyme, it has higher stability than a separated enzyme, it can be proliferated and reactivated, and it does not require the feeding of reducing power such as expensive NADH from the outside. Recently, problems such as restriction of the substance transportation speed and disorders in the cell surface layer, which are the major problems of the use of the whole cell, are being resolved by the development of the technique for surface display localizing the enzyme on the surface of microbial cells.
Prior art techniques for immobilizing microbial cells include entrapment in gel matrix, crosslinking, covalent bonding to a solid surface, and physical adsorption. The used gel, which has been most frequently used, has problems such as the restriction of the substance transportation speed in the gel, cell leakage from the gel, and fragility of the gel. The crosslinking and covalent bonding have problems such as inhibition by the crosslinking agent, and inactivation of cells by the bonding itself. The physical adsorption does not have sufficient adhesiveness for effectively immobilizing general microbial cells, and is only effective for some filamentous fungi. Recently, methodologies for using biofilm as a natural immobilization method are reported (Non-Patent Documents 1 to 8), but there is no method other than screening of the microorganism having the biofilm forming capacity and desired reaction activity, and versatility of the type of microorganisms and reactions is low. In addition, the method is not efficient because it depends on naturally formed biofilm, and is not on the level applicable to actual substance production. Accordingly, prior art immobilization methods are not truly effective, and have many problems, so that the development of a general-purpose and effective immobilization method has been desired.
Acinetobacter sp. Tol 5 (Acinetobacter sp. Bacterium, Tol 5 strain), which was isolated from a biofilter by the inventors, is a nonpathogenic gram negative bacterium which has high autoagglutinating properties of cells, and shows high adhesiveness to various material surfaces such as various hydrophobic plastic carriers and hydrophilic glass and metal surfaces. As a factor giving such adhesive properties which is not reported for other microorganisms, novel bacterionanofiber existing on the bacterial cell surface layer was discovered, and a new protein composing the nanofiber was identified. This protein belongs to the trimeric autotransporter adhesin (TAA) family, and the present inventors named it AtaA (Non-Patent Document 9). TAA is known as a pathogenic factor included in various gram-negative pathogenic bacteria for specifically adhering to the host cells and extracellular matrix such as collagen, fibronectin, and laminin, thereby infecting the host (Non-Patent Document 10). The protein belonging to the TAA family forms a homotrimer, and builds a common whole structure of head-stoke-membrane anchor domain from the amino terminal toward the carboxyl terminal. However, there are small to large single peptide chains having about 300 to over 3000 amino acid residues, and the amino acid sequences are diversified. The peptide chain of AtaA found by the inventors is composed of 3630 amino acids, and is one of the largest TAAs. It has a unique primary structure composed of a plurality of long repeated sequences arranged in a mosaic pattern on a long stoke. Only the AtaA exhibits nonspecific and high adhesiveness to various surfaces. In addition, the study of TAA focuses on pathogenic bacteria, and there is no study on TAA regarding nonpathogenic bacteria such as Tol 5. On the basis of the results of these studies, the inventors reported the method for imparting or enhancing non-specific adhesiveness and/or autoagglutination to the target microorganism through the introduction of the gene encoding AtaA (Patent Document 1). In Patent Document 1, AtaA and the gene encoding AtaA (ataA gene) were referred to as AadA and aadA gene, respectively.